Antimicrobial Susceptibility and Genomic Profiles of Multidrug-Resistant Staphylococcus aureus from Nasopharynx of Asymptomatic Children in Dhaka, Bangladesh

Children carrying Staphylococcus aureus in their nasopharynx are at a higher risk of contracting systemic infection. Due to lack of sufficient information regarding such carriage, this study was conducted to explore the prevalence, antibiotic susceptibility, and genomic profiles of S. aureus isolated from nasopharyngeal samples of 163 randomly selected asymptomatic Bangladeshi children aged from 5–<15 years. Antibiotic susceptibility pattern and genomic analysis of the samples were conducted using standard microbiological methods and genomic tools. The carriage was confirmed in 44 (27%) children who were mostly well nourished without respiratory symptoms in the last 3 months. Higher carriage was observed among the younger age group (5–<10 years) who completed vaccines for pneumonia (p = 0.002) and influenza (p = 0.004). Among the isolates, 84.1% were multidrug-resistant and 47.5% (n = 40) were methicillin-resistant S. aureus (MRSA). All the isolates (100%) were resistant to cefixime with higher resistance to ampicillin (95.5%) and penicillin (90.9%). Among the three investigated isolates, two were ST80 (ID-1 and ID-52) and one was a novel strain (ID-19) with the presence of aph-Stph, blaI, blaZ, dha1, fosB, lmrS, mepA, norA, and tet38 genes. The current research demonstrates a high incidence of multidrug-resistant S. aureus and reports the first instance of ST80 in asymptomatic children in Bangladesh.


Introduction
The nasopharynx and anterior nares are considered the major habitats of S. aureus [1,2], with nasal transmission observed from 16.8% to 90% of strains [1].When S. aureus is acquired as an asymptomatic nasal carriage, it can gradually turn into a persistent carriage where children are especially susceptible to infection due to the systemic invasion Life 2024, 14, 971 2 of 12 of the pathogen [2].According to a study conducted by Esposito et al., out of the 497 healthy subjects, 264 (53.1%) were carriers of S. aureus, with 195 (39.2%) being nasal carriers.However, the study revealed that only three nasal samples (0.6%) were methicillin-resistant S. aureus (MRSA), indicating that the prevalence of MRSA nasal carriage among healthy individuals is relatively low [3].There are few studies on the prevalence of S. aureus in children.Studies in Nepal and Vietnam found S. aureus in the upper respiratory tract in 15% and 29.8% of children, respectively [4,5].Over the years, S. aureus has developed resistance against penicillin and methicillin, which were once effective treatments for the bacteria in the mid-20th century [6].Despite advancements in antibiotic therapy, MRSA still poses a significant risk to global health.Reports from different studies showed that the MRSA isolates were resistant to ciprofloxacin (88.9%) and erythromycin (72.2%) among the identified cases.It was also found that about 20 to 100% of S. aureus isolates were susceptible to different antibiotics [7,8].In China and other Asian countries, the most prevalent hospital acquired-MRSA clones were ST239 and ST5, while community acquired-MRSA clones were diverse such as ST59, ST338, ST30, ST72, and ST8 [9,10].There were two reports of ST80 MRSA isolates among infected patients in Malaysia [11] and Bangladesh [12].It is well documented that MRSA-ST80 isolate is considered a multidrug-resistant pathogen due to its resistance to more than three classes of antibiotics [13].Microbes and human interaction, environmental factors, and host characteristics, including both innate and adaptive immune responses, can predispose to colonization of S. aureus and can result in opportunistic and occasionally life-threatening infections that raise morbidity, mortality, and healthcare costs [14,15].Therefore, it is important to monitor the strains of S. aureus found in the nasopharyngeal carriage since they may be the main source of bacterial dispersion in the community and the primary reservoir of respiratory infections.Screening in the early years of life could be worthwhile to obtain a more precise estimate of S. aureus circulation.There is a lack of information regarding the prevalence of S. aureus strains among the asymptomatic Bangladeshi children.Therefore, this study was designed to explore the nasopharyngeal carriage of S. aureus and their resistance pattern against available antimicrobial agents as well as the genomic profiles of selected isolates among children of different age groups.

Study Population
This study was carried out between April 2022 and January 2023.Nasopharyngeal samples were collected from 163 asymptomatic, and hence, apparently healthy school children aged between 5 and <15 years from 14 randomly selected schools situated in Dhaka, Bangladesh.Among them, 75 children were from 5-<10 years of age, whereas 88 children were aged from 10-<15 years.Children who suffered from infection or in an unhealthy physical state for swab collection or under antibacterial therapy 15 days before sample collection were excluded from the study.

Sociodemographic Characteristics and Nutritional Status
Sociodemographic characteristics, including socioeconomic status, family size, number of siblings, housing condition, education level, and employment status of parents, were assessed in this present study.Nutritional assessment (height for age Z scores, BMI for age Z scores) of each participant was determined [16,17].Other parameters, including vaccination status, episodes of respiratory symptoms, and antibiotic consumption history, were also evaluated.

Nasopharyngeal Specimen Collection and Identification of Bacterial Isolates
Nasopharyngeal specimens were collected using sterile swabs following standard procedure and then cultured onto mannitol salt agar (MSA) media (HiMedia, Mumbai, India).Bacterial isolates were identified based on their colony morphology and biochemical tests according to standard microbiological methods [18,19].

Whole Genome Analysis
Whole genome analysis was performed on three representative isolates: two were MRSA, and one was methicillin-sensitive S. aureus (MSSA).The isolates for genomic analysis were selected based on their antibiotic sensitivity pattern.The MRSA isolates exhibited resistance to most of the therapeutic class of antibiotics, while the MSSA was susceptible.Both MRSA and MSSA isolates were investigated to obtain valuable insights for comparing their resistant phenotypes and genomic profiles.
DNA extraction was performed using the Qiagen DNeasy Blood & Tissue Kit (250) with modification, and the quality of the extracted DNA was evaluated for suitability for subsequent whole genome sequencing (WGS) using both Nanodrop and Qubit measurements.DNA purification was carried out by a modified version of the CDC PULSENET protocol [12].For library preparation, the Illumina DNA Prep Reagent Kit and an automated liquid handler (epMotion 5075) were utilized.The prepared DNA libraries were subjected to sequencing using the Illumina NextSeq550 platform.Sequencing was performed with paired-end 2 × 150 bp reads, yielding comprehensive genetic information.The Phoenix pipeline analyzed raw paired-end reads from the NextSeq550 platform (https://github.com/CDCgov/phoenix)(accessed on 25 February 2024).Species identification was carried out to confirm the organism from which the genomic data were derived.Subsequently, the data underwent a trimming process to remove any adapters and low-quality sequences, employing tools such as BBDuk (https://github.com/BioInfoTools/BBMap)(accessed on 25 February 2024) and Fastp (https://github.com/OpenGene/fastp)(accessed on 25 February 2024).The high-quality, trimmed reads were assembled using the SPAdes [23] algorithm.Followed by sequence typing, antimicrobial resistance (AR) genes were identified utilizing the tools available in AR Gene Curated Databases, such as Resfinder, NCBI, and ARG-ANNOT.

Statistical Analysis
The data were analyzed using Stata software (V.15,StataCorp, College Station, TX, USA).Descriptive statistics, including frequency and proportions, were used to summarize categorical variables, while mean and standard deviation (SD) or median with interquartile range (IQR) were used for quantitative variables.The association between categorical variables was assessed using the Chi-square test or Fisher's exact test, depending on the sample size.For quantitative variables, the association was determined using either the t-test or Mann-Whitney U test, depending on the data distribution.A p-value of less than 0.05 was considered as a statistically significant value.

Ethics Statement
This study was approved by the East West University Research Ethical Committee (Ethical Clearance no.EWUCRT-REC-3/2019) and was conducted in compliance with the Declaration of Helsinki.Informed written consent was obtained from the parents/guardians of all participants before conducting the study.

S. aureus Carriage, Patient Demography, Vaccination and Antibiotic Use Profiles
From a total of 163 healthy children, 66 (40.5%) were male and 97 (59.5%) were female.Nasopharyngeal swab samples of 44 (27%) participants were confirmed for S. aureus carriage with an equal distribution between the two age groups (5-<10 years and 10-<15 years).S. aureus carriage was significantly higher in male compared to the female (p = 0.01).The monthly family income, education, and occupation of the parents indicated that they were from lower middle-class families [24,25].Table 1 shows that the majority of the S. aureus carriers were well nourished children (86.4%) in terms of both BMIZ values and stunting.Among S. aureus carriers from the 5-<10 years age group, about 95.2% of children have completed their EPI vaccination (p < 0.001), 90.5% received the Hib vaccine (p = 0.004), and 85.7% received the PCV vaccine (p = 0.002), which were significantly higher than children from the 10-<15 years age group.Moreover, a larger proportion of younger carriers had respiratory episodes within the last 3 months of the study than the older ones (p = 0.031) (Table 1).

Staphylococcus
showed resistance against six or more antibiotics.About 47.5% (n = 40) of S. aureus isolates showed resistance to cefoxitin, and hence, considered as MRSA.Common resistance phenotypes among the multidrug-resistant isolates are shown in Table 3.

Whole Genome Analysis and Phenotype-Genotype Correlation
The genome quality and contents of the organisms specified were satisfactory, as revealed by the Phoenix output.Overall, the quality of the isolates was also satisfactory, indicating a higher coverage depth of the genomes, ranging from 70× to 130×.The assembly ratio of the genome, which is the ratio between the total number of bases in the sample assembly compared to the expected genome size, was less than 2.58.The number of scaffolds found was also less than 50, indicating a smaller gap and much better genome quality.Additionally, the GC content (32%) was also very satisfactory.The taxonomic identification process of Phoenix is carried out by three tools, namely, MLST, ANI Common resistance phenotypes among the multidrug-resistant isolates are shown in Table 3.

Whole Genome Analysis and Phenotype-Genotype Correlation
The genome quality and contents of the organisms specified were satisfactory, as revealed by the Phoenix output.Overall, the quality of the isolates was also satisfactory, indicating a higher coverage depth of the genomes, ranging from 70× to 130×.The assembly ratio of the genome, which is the ratio between the total number of bases in the sample assembly compared to the expected genome size, was less than 2.58.The number of scaffolds found was also less than 50, indicating a smaller gap and much better genome quality.Additionally, the GC content (32%) was also very satisfactory.The taxonomic identification process of Phoenix is carried out by three tools, namely, MLST, ANI (Average Nucleotide Identity), and Kmerfinder.However, the MLST result shows sequence type of ST 80 for ID-1 and ID-52 and Novel allele type is indicated for ID-19.With the help of the Kraken tool, only one genus (confirmed as S. aureus) has been found above the 25% threshold, which indicates no contamination of other species.
Genotype analysis of three isolates of S. aureus (ID-1, ID-19, and ID-52) revealed that they possessed six antimicrobial resistance (AMR) genes-aph-Stph, dha1, norA, tet38, mepA, and lmrs-with corresponding resistance to aminoglycosides, phenicols (chloramphenicol), quinolones, tetracyclines (tetracycline and tigecycline), and lincosamides, respectively (Table 4).The ID-1 and ID-52 isolates also contained beta-lactam resistance genes mecA (methicillin), blaI and blaZ (penicillin), and macrolide and lincosamide resistance gene ermC.Moreover, these two isolates showed resistance to fluoroquinolone (ciprofloxacin and levofloxacin) due to the presence of two point mutations, one in gyrA_S84L and the other in parC_S80F, in the quinolone-resistance determining regions (QRDRs) of isolates.They were also characterized by the presence of four different plasmid replicons-rep7c, rep5a (pN315), rep10 (pDLK1), and rep16 (pSaa6159).The isolate ID-19, however, exclusively harbored the fosB gene that is known to confer resistance against fosfomycin (Table 4).Findings of the whole genome sequencing, which confirmed ID-1 and ID-52 as MRSA and ID-19 as MSSA corroborate with phenotypic results based on cefoxitin sensitivity as obtained from the VITEK ® 2 data.

Discussion
Asymptomatic colonization of S. aureus increases the risk of invasive infections.To our knowledge, this is the very first report from Bangladesh on the nasopharyngeal carriage of S. aureus and their resistance pattern in healthy, asymptomatic children.
In the current study, about 27% of nasopharyngeal samples tested positive for S. aureus, with 84.1% showing MDR phenotype.Similar to our study, Awulachew et al. reported a global nasopharyngeal carriage of S. aureus (25%) in children between the ages of 6 and 15 [26].According to a cross-sectional investigation, 10.3% of all samples (n = 234), 24 positive cultures contained S. aureus in children aged ≤10 years, with a higher carriage rate of 14.3% between 8 and 10 years [27].In our study, S. aureus carriage was significantly higher in male children than in females.This difference may be attributed to the variation in sex chromosomes.Females possess two X chromosomes, each enriched with 1100 genes and harboring several immune function-regulating genes.On the contrary, males having single X chromosomes make them more vulnerable to infection.Additionally, lifestyle factors, exposure to risk factors, and healthcare-seeking behaviors may also contribute to differentiating the carriage rate of S. aureus between male and female children [28].Our research also revealed that S. aureus carriage is higher among the children who received PCV and Hib vaccines.The introduction of PCV-13 might have contributed to the rise in S. aureus prevalence in the nasopharynx [29].
In a sub-group analysis of 40 multidrug-resistant isolates of S. aureus, we found that 47.5% of the isolates were MRSA.Similar results were seen in other studies where the subjects were MRSA carriers [30,31]; however, the carriage rate was lower in those studies than ours.We found 100% cefixime-resistant S. aureus in this present study, which, according to our knowledge, has not been reported to date.Different studies demonstrated cefixime resistance in patients against S. aureus isolates in Bangladesh.One study demonstrated 80% and 85.2% resistance of the isolates to cefixime from urine samples in males and females, respectively [32].
According to various studies, S. aureus resistance to erythromycin in school children ranged from 20 to 46% [4,33].A study from Bangladesh also demonstrated that less than 40% of isolates were sensitive to erythromycin and azithromycin [7].In our investigation, however, S. aureus exhibited comparatively higher macrolide resistance (63.3% for azithromycin and 68.2% for erythromycin).
All three investigated isolates harbored both tet38 and mepA genes, which are linked to the antibiotic efflux mechanism; however, isolates ID-1 and ID-52 were found to be sensitive to tetracycline and tigecycline, respectively, in phenotype analysis.A study carried out by Zeng et.al., reported that the active tetK efflux pump was primarily responsible for tetracycline resistance [36].This study also observed mutation of the mepA gene and premature termination of the respective efflux protein in tetracycline-sensitive isolates.Moreover, low expression of tet38 and mepA genes was also responsible for tetracycline sensitivity [36].The mepA protein pump is regulated by mepR, which is a part of mepRAB cluster [37].This explains the sensitivity profile of investigated isolates for tetracyclines and other antibiotics (aminoglycosides and chloramphenicol) in the present study.
Plasmids bear mobile genetic elements (MGEs) and are the major means of horizontal transfer of resistance determinants and virulence factors [38].Isolates ID-1 and ID-52 were characterized by the presence of four different plasmid replicons-rep7c, rep5a (pN315), rep10 (pDLK1), and rep16 (pSaa6159).The rep5a was reported as a common plasmid replicon in Malaysian MRSA isolates [29], which is similar to our findings.Moreover, the acquisition of rep10 and rep16 has been found to be responsible for macrolide and lincosamide, and beta-lactam antibiotic resistance, as rep10 and rep16 plasmid replicons harbored ermC and blaZ genes, respectively, and thus have been implicated in the transmission of genetic information among species [29,39].In Central China, pork samples were reported to contain S. aureus harboring rep10 and rep16 [40].The presence of these resistant genes might be useful in understanding the spread of resistant pathogens in Bangladesh, as found in other Asian countries.Therefore, understanding the distribution and function of MGEs and plasmids in S. aureus is crucial for combating antibiotic resistance.This understanding can aid in monitoring MGEs to track the spread of resistance, while genomic analyses provide a comprehensive view of resistance scenarios.Additionally, studying the mechanisms of MGEs and plasmids enables researchers to predict resistance trends, design targeted interventions, and develop strategies to prevent gene transfer.Practical strategies may include infection control, antibiotic stewardship, and research into novel treatments.Overall, this understanding is essential for devising effective measures to manage and mitigate antibiotic resistance.
A unique aspect of this study is detecting antibiotic-resistant microorganisms in nasopharyngeal samples obtained from asymptomatic school children.It includes a wide range of variables to better understand the potential connections to antimicrobial resistance in apparently healthy children, which is a gap in current research.Additionally, previous studies have not thoroughly examined various schools in Dhaka city in a single publication to comprehend patterns of antimicrobial resistance.By involving children from 14 schools in Dhaka city, our study underscores its scientific robustness.While we acknowledge that the sample population may not perfectly represent the entire nation, we are confident in our findings regarding the nasopharyngeal carriage of S. aureus.However, further studies are needed with data collected from various districts of Bangladesh to gain a more comprehensive understanding of the prevailing situation.

Conclusions
In our study, we detected the presence of multidrug-resistant ST80 MRSA isolates, which is, to our best knowledge, the first report from Bangladesh among asymptomatic children.S. aureus is prevalent in nasopharyngeal carriage among school children in Dhaka, and most of these isolates were multidrug-resistant.MRSA was also detected in these samples.One of the reasons for multidrug resistance may be the indiscriminate use of antibiotics among children.It is essential for the nationwide antimicrobial resistance (AMR) surveillance program to consistently monitor antimicrobial resistance to S. aureus in the population, including children.Promoting the responsible use of antibiotics across the nation, implementing routine screening for AMR, and ensuring that policymakers take necessary actions to control the issue are crucial strategies.Public awareness of antibiotic usage restrictions should also be implemented.
The present study on the antimicrobial susceptibility and genomic profile of Staphylococcus aureus nasal carriage in children in Dhaka, Bangladesh, provides valuable insights into understanding the prevalence of this bacterium and its susceptibility to antimicrobial agents.Continuing a thorough longitudinal study with a substantial sample size is crucial to validate the findings.This approach will allow us to effectively integrate our research into comprehending the modes of contamination and transmission of bacteria, especially in identifying S. aureus in siblings and school groups.Moreover, exploring the prevalence and diversity of MGEs in larger and more diverse S. aureus strains and conducting functional studies on the identified plasmid replicons and their encoded resistance genes will provide deeper insights into their roles in resistance and virulence.The outcomes can then be utilized to formulate effective prevention and treatment strategies to tackle the issue.

Figure 1 .
Figure 1.Antibiotic resistance pattern of S. aureus among different age groups.R0: no antibiotic resistance; R1 to R5: resistance to 1 antibiotic to 5 antibiotics; and >R5: resistance to 6 to 14 antibiotics; MDR: resistance to 3 or more classes of antibiotics.Abbreviation: MDR, multidrug-resistant.

Figure 1 .
Figure 1.Antibiotic resistance pattern of S. aureus among different age groups.R0: no antibiotic resistance; R1 to R5: resistance to 1 antibiotic to 5 antibiotics; and >R5: resistance to 6 to 14 antibiotics; MDR: resistance to 3 or more classes of antibiotics.Abbreviation: MDR, multidrug-resistant.

Table 1 .
Age group-wise distribution of S. aureus carriers.

Table 1 .
Cont.Data are presented as number, n (%).BMI is represented as mean ± SD, whereas family size, number of siblings, persons living per room, and family income are presented as median with interquartile range (IQR).a Fisher's exact test; b Chi-square test; c Student's t-test; d Mann-Whitney U test.Abbreviations: BMI, body mass index; BMIZ, body mass index for age; EPI, expanded program on immunization; Hib, Haemophilus influenzae type b; PCV, pneumococcal conjugate vaccine.

Table 2 .
Age group-wise distribution of antibiotic-resistant S. aureus and MRSA.

Table 4 .
Antimicrobial-resistant phenotypes and genotypes of selected samples of S. aureus.